Picornaviruses, vaccines and diagnostic kits

ABSTRACT

(I)                   SEQ ID NO: 1 (Ljungan 87-012)         AGTCTAGTCT TATCTTGTAT GTGTCCTGCA CTGAACTTGT TTCTGT               CTCT 50                          GGAGTGCTCT ACACTTCAGT AGGGGCTGTA CCCGGGCGGT CCCACT                   CTTC 100                         ACAGGAATCT GCACAGGTGG CTTTCACCTC TGGACAGTGC ATTCCA                   CACC 150                         CGCTCCACGG TAGAAGATGA TGTGTGTCTT TGCTTGTGAA AAGCTT                   GTGA 200                         AAATCGTGTG TAGGCGTAGC GGCTACTTGA GTGCCAGCGG ATTACC                   CCTA 250                         GTGGTAACAC TAGC                                                                                          
A new group of picornaviruses is disclosed. The picornaviruses of the invention comprise in the non-coding region of their viral genome a nucleotide sequence which corresponds to cDNA sequence (1) or homologous sequences having at least 75% homology to the SEQ ID NO:1, and they cause mammalian disease. Further aspects of the invention comprise a protein corresponding to a protein of the picornaviruses, antiserum or antibody directed against a protein of the picornaviruses, antigen comprising a protein of the picornaviruses, diagnostic kits, vaccines, use of the picornaviruses in medicaments, particularly for the treatment or prevention of Myocarditis, Cardiomyopathia, Guillain Barré Syndrome, and Diabetes Mellitus, Multiple Sclerosis, Chronic Fatigue Syndrome, Myasthenia Gravis, Amyothrophic Lateral Sclerosis, Dermatomyositis, Polymyositis, Spontaneous Abortion, and Sudden Infant Death Syndrome, and methods of treatment of diseases caused by the picornaviruses.

FIELD OF INVENTION

The present invention relates to new picornaviruses, proteins expressedby the viruses, antisera and antibodies directed against said viruses,antigens comprising structural proteins of said viruses, diagnostickits, vaccines, use of said viruses, antisera or antibodies and antigensin medicaments, and methods of treating or preventing diseases caused bysaid viruses, such as Myocarditis, Cardiomyopathia, Guillain BarréSyndrome, and Diabetes Mellitus, Multiple Sclerosis, Chronic FatigueSyndrome, Myasthenia Gravis, Amyothrophic Lateral Sclerosis,Dermatomyositis, Polymyositis, Spontaneous Abortion, and Sudden InfantDeath Syndrome.

BACKGROUND OF THE INVENTION

Recently, a sudden death syndrome among Swedish orienteers has beenobserved. Of approximately 200 elite orienteers six died in myocarditisduring 1989-1992 (1). Orienteering, aiming to find the fastest/shortestway between several checkpoints and often in forested areas, isexceptional with respect to environmental exposure. Thus it has beenspeculated, that the sudden deaths syndrome among orienteers is causedby a vector borne (rodent or arthropod) infectious agent.

It has now been shown in an epidemiological study that the incidence ofdeaths in myocarditis in northern Sweden tracked the 3-4 year populationfluctuations (cycles) of bank voles (Clethrionomys glareolus) with oneyear time lag. Previously, it has been shown that cardioviruses, withrodents as their natural reservoir, can cause Guillain Barré Syndrome(GBS) in man, Diabetes Mellitus (DM) in mice and myocarditis in severalspecies including non-human primates.

In addition to death in myocarditis it is also shown in theepidemiological study that the number of patients diagnosed withGuillain Barré Syndrome (GBS), and Diabetes Mellitus (DM) in northernSweden tracked the 3-4 year population fluctuations of bank voles withdifferent time delays.

Sven Gard and co-workers studied antibody prevalence toencephalomyelitis virus (EMCV) in Swedish normal population in the early1950th (2). These studies found a surprisingly high antibody prevalencerate by hemagglutination inhibition test but no sera could be confirmedby neutralization test. These results were found puzzling at the timebut could be explained by the presence of one or several relatedpicornaviruses circulating in Sweden.

The fact that enterovirus have a large number of members and cardiovirusonly two possibly three could reflect the true diversity of the twogenus or only be the result of the amount of effort made to isolate newviruses from rodents as compared to isolating new enteroviruses fromhumans.

The Picornavirus family is presently divided into five genera (aphto-,entero-, hepato-, rhino-, and cardioviruses) (3). This taxonomy wasinitially based on morphological, physiological and serologicalproperties as well as on the pathogenicity of the viruses. Morerecently, however, viruses have been characterized based on their genomesequence since it has been established that sequence data to a largeextent coincide with the characterisation properties used previously(4,5).

The prototype virus in the cardiovirus genus is Theiler's murineencephalomyelitis virus (TMEV). Another member in this genus isencephalomyocarditis virus (EMCV). Vilyuisk virus, isolated frompatients in Russia with degenerative neurological disease, isserologically related to TMEV but presently under consideration forbeing included as a third distinct member of the cardiovirus genus (6).

In nature, cardioviruses have a geographically widespread distributionand a large number of susceptible hosts with rodents as their naturalreservoir. In addition to rodents, EMCV has been isolated from domesticpigs, elephants, lions, non human primates and man (7,8,9). Infectionwith TMEV and EMCV have provided excellent animal models for inducingmyocarditis, DM and different neurological disorders such asdemyelinating diseases resembling multiple sclerosis in mice (10-16).Other neurological or muscular disorders in which an infection issuspected to be the triggering factor and in which there is also anautoimmune component are Cardiomyopathia, Multiple Sclerosis (MS),Chronic Fatigue Syndrome (CFS), Myasthenia Gravis (MG), and AmyothrophicLateral Sclerosis (ALS). It has never been established, however, thatcardiovirus is a significant human pathogen, as disease in man mostoften has been described in case reports or as infection measured insero-epidemiological surveys (7-17).

Thus, there may be other not yet identified picornaviruses circulatingin the wild rodent population and occasionally infecting humansresulting in Myocarditis, Cardiomyopathia, Guillain Barré Syndrome, andDiabetes Mellitus, Multiple Sclerosis, Chronic Fatigue Syndrome,Myasthenia Gravis, Amyothrophic Lateral Sclerosis, Dermatomyositis,Polymyositis, Spontaneous Abortion, and Sudden Infant Death Syndrome, ingenetically susceptible individuals.

The epidemiological link between important human diseases and smallrodent abundance and what is previously known aboutpicornavirus/cardiovirus motivated attempts to isolate novelpicornaviruses from small rodents.

DESCRIPTION OF EXPERIMENTAL WORK AS BASIS FOR THE INVENTION

Trapping of Animals

Small rodents were trapped at several locations in northern Sweden andtransported live to the Swedish Institute for Infectious Disease Controlin Stockholm, Sweden. Species, date and location of trapped animals wererecorded. Animals were bled using ether anaesthesia and killed. Organswere immediately removed and stored at −70° C. until tested for presenceof virus.

A total of 53 Clethrionomys glareolus and 28 Microtus agrestis weretested for virus isolation.

Virus Isolation

The isolation technique used in the present study was different fromwhat is most often used. The cells used for isolation were kept for aminimum of two weeks and virus growths were detected by both CPE(cytopathogenic effect) and by staining the cells by a large number ofhuman sera using IFT (immunofluorescense test). None of the new virusespresented herein would have been isolated using routine procedure fordetecting cardioviruses/picornaviruses. They grow to lower titer and CPEdevelops slowly.

Saliva mixed with lung homogenate and faeces were analyzed separatelyfrom each animal. The material was inoculated into T25 flask ofconfluent BHK-21 cells. Cells were blind passed twice a week during twoweeks. At the end of this period or earlier if signs of CPE occurred,cells were removed from the T25 flask by a rubber policeman, placed onto10-well spot slides, air dried and acetone fixed. The cells were thenstained with panels of human sera including 5 multiple sclerosespatients, 5 patients recently diagnosed with DM and 5 athletes dying inmyocarditis and bled at autopsy. All T25 flasks (saliva-lung and faecesseparately) were tested individually by IFT using the complete panel ofhuman sera at a 1:10 dilution.

Cells showing positive reaction by IFT using the human serum panels wereselected for further analysis. This included inoculation intracerebrallyinto 1 day old suckling mice, serological characterisation and sequenceanalysis.

Antisera and Serological Procedures

Antisera to the virus isolates were raised in mice (NMRI), and GuineaPigs (Dunkin Hartley). The animals were injected with a cell culturesupernatant from (BHK-21 cells) intraperitoneally and serum collected 46weeks later. Preimmunization sera were tested individually whilepostimmunization sera were pooled from all infected animals.

An indirect immunoflourescense test (IFT), as described previously(18,19) was used to test antibody titres in immunized animals. Briefly,spot slides were prepared by incubating virus on Green Monkey Kidney(GMK) cells for 6-10 days. At sign of discrete CPE cells were removedfrom the flask by a rubber policeman and put onto the microscope slides,air dried, and fixed in cold (4° C.) acetone and stored at −70° C. Thetiter was determined by incubating serum diluted in PBS in the slides at37° C. for 1 hour in a moist chamber, followed by a FITC conjugate(F(ab′)₂ fragment of goat anti human IgG γ-chain specific, Sigma ImmunoChemicals (F-1641) or Rabbit anti mouse immunoglobulins Daco (F0313))incubated as above.

Antibody titers to the viruses were determined by a modification of thePlaque reduction neutralization test (PRNT) as described by Earley et al(20). In the test, sera were serially diluted four-fold and mixed withan equal volume containing 80-100 plaque-forming units (pfu) of virusper 50 μl. The mixtures were then incubated at 37° C. for 60 minutes,and 50 μl subsequently inoculated into each of 2 wells of a tissueculture plate containing confluent Vero cell monolayer. After adsorptionfor 60 minutes at 37° C. the wells were overlaid with 0.5 ml of a 42° C.mixture of 1 part 1% agarose and 1 part 2× basal Eagle's medium withEarle's salts, 17 mM Hepes buffer, 8% heated fetal calf serum, 100 U/mlpenicillin, 100 μg streptomycin. The tissue culture plates wereincubated at 37° C. in a humidified 5% CO₂ atmosphere for 3-7 days. Asecond overlay (0.5 ml) containing neutral red stain (1:9000) was thenapplied and plaques were enumerated the following day. The plaquenumbers were linearly extrapolated to 2-fold dilutions. An 50% reductionof plaques was used as the criterion for virus neutralization titers.

Electron Microscopy

Cell culture media or brain tissue homogenates were examined by negativecontrast electron microscopy (EM). A 10 μl droplet was incubated onFormvar/carbon-coated grids for one minute or alternatively, 0.5 mlsamples were centrifuged for 30 minutes at 20,000×g to remove celldebris and finally the supernatants were pelleted directly onto grids ina Beckman Airfuge for 10 minutes at 160,000×g. Grids were stained with2% phosphotungstate acid (pH 6.0) and examined in a Philips CM 100electron microscope at a magnification of at least 46,000.

Sequence Data

The isolates 87-012, 174F and 145SL were grown on the human lungcarcinoma line A549 in 1600 cm² roller bottles. Full CPE was observedafter 5-10 days. Supernate was filtered through 0.45 μM celluloseacetate filters (Costar) and the virus was pelleted at 20,000 g for 20 hat 4° C. RNA was isolated from the virus containing pellets using acidguanidinium thiocyanate as described (Chomczynski and Sacchi). Synthesisof cDNA was performed under standard conditions using 1 μg of RNA, AMVreverse transcriptase (Boehringer-Mannheim) and random 14 meroligonucleotides as primers in a 20 μL reaction. Fragments of the viral5′UTR were amplified using cardiovirus specific consensus primers:(sense) 5′-GGCCGAAGCCGCTTGGAATA-3′ (SEM) and (antisense)5′-GTGGCTTTTGGCCGCAGAG-3′ (ATVEM), both primers modified after the EMCV2and EMCV1 primers previously reported (Jongen et al. 1993. Ann. Reum.Dis. 52:575-578. Cardiovirus sequences were from Dr A. Palmenberg(personal communication). Amplification conditions were 30 cycles at 94°C., 30 sec., 50° C., 30 sec, 72° C., 2 min. The amplified fragments werecloned into the pCRII T-vector (In-Vitrogen). The cloned viral sequenceswere sequenced using A Taq polymerase FS cycle sequencing kit and datawas collected on a ABI Prism 310 sequencing machine using M13-21 and M13reverse primers (Perkin-Elmer). A 1.8 kb fragment extending from the5′-UTR into the viral polyprotein sequences was obtained by PCR(polymerase chain reaction) amplification of cDNA from the 145SLisolate. The primers were: (sense) 5′-ACAGTGCATTCCACAC-3′ (SLJU1) or5′-CCGCTCCACAATAGA-3′ (SLJU2) and (antisense) 5′-GATCTCAGAC-3′ (primer118). The SLJU1 and SLJU2 primers are located immediately adjacent toone another and were chosen as consensus primers for the Ljunganisolates of the invention with as little homology as possible to theEMCV and TMEV groups of viruses. The amplification conditions were 30cycles at: 94° C., 30 sec., 42° C., 1 min, 72° C. 2 min. The antisenseprimer 118 yielded similarly sized PCR products with either the SLJU1 orSLJU2 as sense primers, but none of the primers yielded PCR fragmentswhen used alone. The sequence of the primer 118 was previously published(Bauer, D., et al. 1993. Nucl. Acids Res. 21:4272-4280). The obtained1.8 kb PCR fragment was cloned and sequenced as described above.

Results of Experimental Work

Three virus isolates were selected based on reaction with the humanserum panels and showing a size and structure compatible with apicornavirus on EM. The first isolate was named Ljungan 87-012. Ljunganis a river in Medelpad county, Sweden where the animals were trapped.

The second and third isolate were designated Ljungan 174F and Ljungan145SL, respectively.

All three isolates came from C. glareolus.

All three isolates killed suckling mice in 3-5 days.

The titer in mouse brain was 10⁸ (approximately) while the cell culturetiter was only 10⁵ (approximately).

Electron Microscopy

Virus particles, 27 nm in diameter, were spherical with the surfacealmost featureless and they appeared single or in small aggregates. Inrare cases the stain penetrated the particles which made them look likeempty shells.

Serological Results

It was found after testing a number of different cell lines the GreenMonkey Kidney cells were most suitable for making IFT drop slides forserology. The cross IFT data using mouse sera are seen in Table 1. TABLE1 Cross-IFT using virus infected GMK cells. Immune mice were titratedusing 4 fold dilutions starting at a 1:10 dilution. VIRUS Antisera87-012 174F 145SL 87-012 2560 160 <10 174F 160 160 <10 145SL 40 40 640

PRNT (plaque reduction neutralization test) data, preliminary results.Rabbit sera against TEMV and EMCV with a titer of 1:160 homologous had atiter less than 10 to the three isolates. Several attempts to makeantisera with neutralizing titer in bank voles, mice, rabbits and guineapigs have failed. All animals made high titer antibodies by IFT but noby PRNT. Bank voles failed to make IFT antibodies.

Sequence Data

Sequences from 5′UTR and polyprotein gene of Ljungan virus isolates.

Cardiovirus consensus primers yielded a product of 303 bp for the threeisolates 87-012, 174F and 145SL, compared to 284 bp for EMC virus. Thefragment amplified was located immediately after the end of the poly Ctract in EMC virus. PCR products specific for the Ljungan isolates wereonly obtained when the reannealing temperature was 50° C., and not at58° C., which was optimal for obtaining products from EMC virus cDNA.The subsequent sequence analysis revealed that the ATVEM primer wasmismatched at 4 internal positions, explaining this difference inreannealing temperature. An alignment of the 5′UTR sequences for thethree Ljungan isolates, EMCV and Vilyuisk virus (Table 2) shows agreater similarity between EMCV and Vilyuisk virus than between eitherof the two and the Ljungan isolates. It also demonstrates that eachLjungan isolate is distinct from the other by a number of nucleotidechanges. The 174F and 145SL are similar to the isolate 87-012. Thesequence homology between 174F and 87-012 was at most 95% (threeundetermined bases in the sequence) while the homology between 87-012and 145SL was 91%.

The strategy chosen for obtaining additional PCR fragments from theLjungan virus isolates was a modification of a technique for detectingdifferentially expressed mRNAs (Bauer, D., et al. 1993. Nucl. Acids Res.21:4272-4280). As a test for this strategy, cDNA from the Ljungan 145SLisolate was amplified using the conditions above, using either the SLJU1or the SLJU2 primer as a sense primer and one of twenty 10-meroligonucleotides of randomly chosen sequence as “antisense” primer.

If the PCR products obtained with the SLJU1 or SLJU2 primers and aspecific 10-mer were similarly sized, and none of the primers yielded aproduct of this size when used alone in the PCR reaction, the fragmentobtained was isolated and cloned. Only one combination of primerssatisfied this criterion, namely the SLJU1 or SLJU2 primers incombination with the 118 10-mer oligonucleotide, which yielded a 1.8-1.9kb PCR product. Of this fragment, 819 bp were sequenced from the 3′ end.This sequence contained an open reading frame (ORF) of 663 bp in thesense of the viral polyprotein. This ORF was used to search in the Swissprotein data bank using the BLITZ search service from EMBL with thedefault search parameters. The top 10 scores were picornaviruspolyprotein sequences, including 8 cardiovirus sequences. Homology wasfound over 188 a.a. The relatedness of this segment of the viralpolyprotein to previously sequenced cardioviruses is shown in Table 3. Acomparative alignment of all cardioviruses was made available to us byDr. A. Palmenberg. In Table 3, the sequence of TMEBeAn was arbitrarilytaken as the index strain. For the 12 remaining cardioviruses in thealignment, only differences in amino acid sequence are shown. Thealignment of the Ljungan 145SL sequence is similarly represented at thetop. Since the BLITZ search algorithm takes into account identical aswell as similar amino acids, the latter have been indicated by smalltype, while differences to TMEBeAn is in capitals as for the otherstrains in the alignment.

In conclusion, the above presented data for the Ljungan isolates arecharacteristic for the 3 viruses but yet incomplete. However, thecomparison of cloned sequences from both a highly conserved part of the5′-untranslated region of cardioviruses and coding sequences for theviral capsid proteins of one isolate (Ljungan 145SL) clearly show thatthe Ljungan viruses are related to the cardioviruses, but are moredistant relatives than any previously identified cardiovirus. While theamino acid homology (identical amino acids) of the viruses within theTheiler group is 96-97%, the homology to Vilyuisk virus is about 83%,and the EMC viruses are 67-74% homologous to TMEBeAn, the Ljungan 145SLhas only about 32% identical amino acids to TMEBeAn. Even if homology istaken as identical and similar amino acids, this measure of relationshipwould still amount to only 50% between Ljungan 145SL and TMEBeAn (thecorresponding figure would be 79-83% between EMC and TMEBeAn).

Alignment of Sequences

Table-2 shows an alignment of three Ljungan virus isolates (1.87-012, 2.174F, 3. 145SL)[SEQ ID NO: 1,2 and 3, respectively] with publishedcardiovirus sequences (4. TMEBeAn, 5. Vilyuisk, 6. EMCV). The alignedsequence starts 29 nt 3′ of the end of the poly-C tract in EMCV, and thesequence corresponds to nt 557-808 (approximately) in the differentviral genomes. Inserted spaces in the sequences are indicated by aperiod (.). TABLE 2 1.AGTCTAGTCTTATCTTGTATGTGTCCTGCACT..GA..ACTTGTTTCTGT 2.AGTCTAGTTTCATTCTGTGTGTGTTTGGCACT..GA..AATTATTTCTGT 3.AGTTTGGTTCTCTCTTGAGTGTGTTTTGTGTT..AG..CATAATTTCTGT 4.TGACAGG.GTTATTTTCACC.TCTTCTT..TTCTACTCCACAG.TG.T.T 5.TGACAGG.GTTATTTTCACC.TCTTCTCTCTTCTACTTCATAG.TG.T.T 6.AGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCAC..CATA.TTGCCGT 1.CTCTGGAGTGCTCTACACTTCAGTAGGGGCTGT.A.CCCGGGCGGTCCCA 2.CTCTGGGGTGCTTTACACTTCAGTAGGGGCTGT.A.CCCGGGCGGTCCCA 3.CTCTAGAGTGCTTTACACTCTAGTAGGGGCTGT.A.CCCGGGCGGTCCCA 4.CT.A.........TACTGTG..GAAGGGTATGTGT....TGCCCCTTCCT 5.CT.A.........TACTATG.AA.AGGGTATGTGT..C..GCCCCTTCCT 6.CT.T.......TTGGCAATGT.G.AGGGCCCG.GAAACCTGGCCCTGTCT 1.CTCTTCACAGGAATCTGCACAGGTGGCTTTCAC.CTCTGGACAGTGCATT 2.CTCTTCACAGGAATNTGCACAGGTGGCTTTCAC.CTCTGGACAGTGCATT 3.CTCTTCACAGGAATCTGCACAGGTGGCTTTCAC.CTCTGGACAGTGCATT 4..TCTTGGAGAACGT..GCGCGGCGGTCTTTCCGTCTCTCGACAA.GCGC. 5..TCTTGGAGAACGT..GCGTGGCGGTCTTTCCGTCTCTCGAAAAACG..T 6..TCTTGACGAGCAT.T.CCTAGGGGTCTTTCCC.CTCTCGCCAAAGGAAT 1.CCACACCCG.C.TCCACGGTAGAAGATGATGTGTGTCTTTGCT..TGTGA 2.CCACACCCG.C.TCCACAGTAGAAGATGATGTGTGTCTTTGCT..TGTGA 3.CCATACCCG.C.TCCACAATAGAAGATGATGTATATCTTTGTT..TGTGA 4.GCGT..GCAACATACAGAGT.AACG.CGAAGAA.AGCA..GTTC.TC.GG 5.GCGT..GCGACATGCAGAGT.AACG.CAAAGAA.AGCA..GTTC.T.TGG 6.GCA.A.G.GTC.TGTTGAAT.GTCG.TGAAGGA.AGCA..GTTCCTCTGG 1.AAA.GCTT...GTGAAAATC........GTGTGTAGGCGTAGCGGCTACT 2.AAA.GCTT...GTGAAAATC........GTGTGTAGGCGTAGCGGNTACT 3.AAT.GCT.CA..TGAA.A.C......GTGTGTGTAGGCGTAGCGGCTACT 4.TCTAGCT.CTAGTGCCCA.CAAGAAAACAGCTGTAG.CG.ACCA.C.ACA 5.TCTAGCT.CTGGTGCCCA.CAAGAAAACAGCTGTAG.CG.ACCA.C.ACA 6.AA..GCTTCT..TGAAGA.CAA.ACAACGTCTGTAG.CG.ACC..CT..T 1.TGAGTGCCAGCGGATTACCCCTAGTGGTAACACTAGC 2.TGAGTGCCAGCGGACNACCCCTAGTGGTAACACTAGC 3.TGAATGCCAGCGGAACCCCCCTAGTGGTAACACTAGC 4...AAGGC.AGCGGAACCCCCCTCCTGGTAACAGGAGC 5...AAGGC.AGCGGAAACCCCCTCCTGGTAACAGGAGC 6.TGCAGGC.AGCGGAACCCCCCACCTGGCGACAGGTGCIn this region of the viral genome, Ljungan 174F has 94% homology toLjungan 87-012 (here taken as the indicator strain for comparisons), andLjungan 145SL has 91% homologous residues to Ljungan 87-012. The TMEBeAnstrain has 69%, Vilyuisk has 68% and EMCV has 68% homologous residues toLjungan 87-012. Using the same criteria for calculating the homology,EMCV has 85% homology to TMEBeAn.

Table 3 shows alignment of cDNA sequences from the polyprotein codingsequences of the Ljungan 145SL isolate [SEQ ID NO. 4] to the amino acidsequences of sequenced cardioviruses in the comparative alignmentcompiled by Dr. A. Palmenberg (personal comm.) The TMEBeAn strain wasarbitrarily taken as the indicator strain, while the amino acids of theremaining strains are shown only if they differ from the indicatorstrain. For the Ljungan 145SL isolate, similar, but non-identical aminoacids are indicated in small type. The amino acid homology betweenLjungan 145SL and other cardioviruses was established screening theentire Swiss Protein Data Bank using the BLITZ search algorithm withstandard search parameters. TABLE 3 Ljungan464            480                                         525 145SLK--m-iARM-sVyK-ERTEPGGTNG--QWshtHSPInW-.fDGGiHLED-P-..-LFsSCy- TMEBeAnSDLLELCKLPT.FLGNPNTNNKRYPYFSATNSVPATSMVDYQVALSCSCMANSMLAAVARNFN TMEGd7-----------.-----S-D-----------------L------------------------- TMEGd7-----------.-----S-D-----------------L------------------------- TMEDa-----------.------S---------------T--L------------C------------ VilyuiskT----------.----L.S-DT-V-F-T------TE-L-E---T------S-----S------ EMCBdK-F--IAQI--.-I--KIP-AVP-IEA-N-.A-KTQPLAT---T-----L--TF---LS---A EMCBcK-F--IAQI--.-I--KIP-AVP-IEA-N-.A-KTQPLAT---T-----L--TF---LS---A EMCDdK-F--IAQI--.-I--KIP-AVP-IEA-N-.A-KTQPLAT---T-----L--TF---LS---A EMCDcK-F--IAQI--.-I--KIP-AVP-IEA-N-.A-KTQPLAT---T-----L--TF---LS---A EMCDv1K-F--IAQI--.-I--KIP-AVP-IEA-N-.A-KTQPLAT---T-----L--TF---LS---A EMCRK-F--IAQI--.-I--KIP-AVP-IEA-N-.A-KTQPLAT---T-----L--TF---LS---A MengoMK-F--IAQI--.-I--KMP-AVP-IEA-N-.A-KTQPLAV---T-----L--TF---LS---A Mengo37aK-F--IAQI--.-I--KVP-AVP-IEA-N-.A-KTQPLAV---T-----L--TF---LS---A Ljungan526         540                                             588 145SLYw---TVLKLTVYAsTFN--rLRm-fF-I.MMqG-Q-.kKHkCLfMvC-i---nt-EM-I-y. TMEBeAnQYRGSLNFLFVFTGAAMVKGKFLIAYTPPGAGKPTTRDQAMQSTYAIWDLGLNSSFNFTAPFI TMEGd7----------------------R-------------------A-------------------- TMEGd7b------------------------------------------A-------------------- TMEDa------------------------------------------A-------------V------ Vilyuisk--------------S--T----------------------X------------------V--- EMCBd------VYT-----T--M-----------------S------A------------YS--V--- EMCBc------VYT-----T--M-----------------S------A------------YS--V--- EMCDd------VYT-----T--M-----------------S------A------------YS--V--- EMCDc------VYT-----T--M-----------------S------A------------YS--V--- EMCDv1------VYT-----T--M-----------------S------A------------YS--V--- EMCR------VYT-----T--M-----------------S------A------------YS--V--- MengoM------VYT-----T--M-----------------S------A------------YS--V--- Mengo37a------VYT-----T--M-----------------S------A------------YS--V--- Ljungan589      600                                                 651 145SL..-.w..GnwMR--RG--I--lRiDV-NR---N-Ss-NAVnCiLQ-KM-n-AKRMv-TT-NIV- TMEBeAnSPTHYRQTSYTSPTITSVDGWVTVWKLTPLTYPSGTPTNSDILTLVSAGDDFTLRMP.ISPTKW TMEGd7-------------------------Q-------------------------------.------ TMEGd7b-------------------------Q-------------------------------.------ TMEDa------------A--A---------Q---------A-V-------------------.------Vilyuisk--S----------S-AA----L---Q-----F-ANV-PS----------N-------.------ EMCBd----F-MVGTDQVN--N--------Q-------P-C--SAK---M----K--S-K--.---AP- EMCBc----F-MVGTDQ-------------Q-------------------------------.------ EMCDd----F-MVGTDQ-------------Q-------------------------------.------ EMCDc----F-MVGTDQ-------------Q-------------------------------.------ EMCDv1----F-MVGTDQ-------------Q-------------------------------.------ EMCR----F-MVGTDQ-----A-------Q-------------------------------.------ MengoM----F-MVGTDQA------------Q-------------------------------.------Mengo37a----F-MVGTDL-----A-------Q-------------------------------.------Serological Assay Indicating Relationship Between the Ljungan Virusesand Diabetes Mellitus and Myocarditis.

A serological assay using indirect immunofluorescense test using virusinfected acetone fixed green monkey kidney cells was established.Patient sera were screened at a 1:8 dilution and positive sera titrated.Sera with a titer of 1:32 or more were considered positive.

Sera from 59 children (age 1-16) from the Stockholm area recentlydiagnosed with Diabetes Mellitus (DM) and 34 control children from thesame geographic area, were tested for presence of antibodies to thethree viruses of the invention. Nineteen of the 59 (32%) DM patientsscreened positive and 2 of the 34 (6%) controls were found positive toone or more of the 3 viruses (significant difference p=0.002, Fisher'sexact test). Nine recently diagnosed DM patients (age 23-46) fromMedelpad county were also tested. Two controls were selected for eachadult DM patient and they were matched for age, sex and geographic areaof residence. Five of the nine (56%) DM patients and one of the 18 (6%)control patients were found positive to one or more of the 3 viruses(significant difference p=0.008 Fisher's exact test).

Serum was also available from 5 athletes dying suddenly in myocarditis.Three controls were selected for each myocarditis patient and they werematched for age, sex and geographic area of residence. Four of the 5(80%) patients dying from myocarditis and 1 of the 15 (7%) controlswhere found positive to one or more of the three Ljungan viruses(significant difference p=0.005, Fisher's exact test).

DESCRIPTION OF DIFFERENT ASPECTS OF THE INVENTION

In the following, different aspects of the invention will be disclosed.However, all of these aspects are related to a new group ofpicornaviruses.

Thus, a first aspect of the invention is directed to a new group ofpicornaviruses, namely picornaviruses comprising in their viral genome,more precisely in the non-coding region, a nucleotide sequencecorresponding to a cDNA sequence selected from the group consisting ofSEQ ID NO: 1 (Ljungan 87-012) AGTCTAGTCT TATCTTGTAT GTGTCCTGCACTGAACTTGT TTCTGTCTCT  50 GGAGTGCTCT ACACTTCAGT AGGGGCTGTA CCCGGGCGGTCCCACTCTTC 100 ACAGGAATCT GCACAGGTGG CTTTCACCTC TGGACAGTGC ATTCCACACC150 CGCTCCACGG TAGAAGATGA TGTGTGTCTT TGCTTGTGAA AAGCTTGTGA 200AAATCGTGTG TAGGCGTAGC GGCTACTTGA GTGCCAGCGG ATTACCCCTA 250 GTGGTAACACTAGCand homologous sequences having at least 75% homology to the SEQ IDNO: 1. The picornaviruses of the invention should further causemammalean disease.

In a preferred embodiment of this aspect of the invention saidhomologous sequences have at least 80%, at least 85% or at least 90%homology to the SEQ ID NO: 1.

In a particularly preferred embodiment said homologous sequence is oneof SEQ ID NO: 2 (Ljungan 174F) AGTCTAGTTT CATTCTGTGT GTGTTTGGCACTGAAATTAT TTCTGTCTCT  50 GGGGTGCTTT ACACTTCAGT AGGGGCTGTA CCCGGGCGGTCCCACTCTTC 100 ACAGGAATNT GCACAGGTGG CTTTCACCTC TGGACAGTGC ATTCCACACC150 CGCTCCACAG TAGAAGATGA TGTGTGTCTT TGCTTGTGAA AAGCTTGTGA 200AAATCGTGTG TAGGCGTAGC GGNTACTTGA GTGCCAGCGG ACNACCCCTA 250 GTGGTAACACTAGC and SEQ ID NO: 3 (Ljungan 145SL). AGTTTGGTTC TCTCTTGAGT GTGTTTTGTGTTAGCATAAT TTCTGTCTCT  50 AGAGTGCTTT ACACTCTAGT AGGGGCTGTA CCCGGGCGGTCCCACTCTTC 100 ACAGGAATCT GCACAGGTGG CTTTCACCTC TGGACAGTGC ATTCCATACC150 CGCTCCACAA TAGAAGATGA TGTATATCTT TGTTTGTGAA ATGCTCATGA 200AACGTGTGTG TAGGCGTAGC GGCTACTTGA ATGCCAGCGG AACCCCCCTA 250 GTGGTAACACTAGC.

These sequences (ID NO: 2 and 3) have 94% homology and 91% homology tothe SEQ ID NO: 1, respectively.

It should be understood that homologies in the coding region ofdifferent viruses of the invention may vary considerably, but in thenon-coding region they share a homology of at least 75% with the SEQ IDNO: 1.

The nucleotide sequences, SEQ ID NO: 1, 2 and 3, correspond toapproximately nucleotides 557-808 (a conserved region) in the genome ofencephalomyelitis virus (EMCV). These three viruses have been isolatedfrom wild rodents, more precisely bank voles. The viruses can bemultiplied in cell lines, and for a large-scale production ofpicornavirus products the virus genome can be inserted into othermicroorganisms.

A second aspect of the invention is directed to a protein comprising anamino acid sequence selected from the group consisting of SEQ ID NO: 4(partial structural protein of Ljungan 145) Lys Asp Leu Met Glu Ile AlaArg Met Pro Ser Val Tyr Lys Gly Glu                 5                  10                  15 Arg Thr GluPro Gly Gly Thr Asn Gly Tyr Phe Gln Trp Ser His Thr            20                  25                  30 His Ser Pro IleAsn Trp Val Phe Asp Gly Gly Ile His Leu Glu Asp        35                  40                  45 Met Pro Asn Leu AsnLeu Phe Ser Ser Cys Tyr Asn Tyr Trp Arg Gly    50                  55                  60 Ser Thr Val Leu Lys LeuThr Val Tyr Ala Ser Thr Phe Asn Lys Gly 65                 70                 75                   80 Arg LeuArg Met Ala Phe Phe Pro Ile Met Met Gln Gly Thr Gln Arg                85                  90                  95 Lys Lys HisLys Cys Leu Phe Met Val Cys Asp Ile Gly Leu Asn Asn            100                 105                 110 Thr Phe Glu MetThr Ile Pro Tyr Thr Trp Gly Asn Trp Met Arg Pro        115                 120                 125 Thr Arg Gly Ser ValIle Gly Trp Leu Arg Ile Asp Val Leu Asn Arg    130                 135                 140 Leu Thr Tyr Asn Ser SerSer Pro Asn Ala Val Asn Cys Ile Leu Gln145                 150                 155                 160 Val LysMet Gly Asn Asp Ala Lys Phe Met Val Pro Thr Thr Ser Asn                165                 170                 175 Ile Val Trp,and homologous sequences having at least 75% homology to the SEQ ID NO:4,

-   and antigenic fragments of the sequences.    In an embodiment of the invention the homologous sequences have at    least 80%, at least 85% or at least 90% homology to the SEQ ID NO:    4.

The SEQ ID NO: 4 is the result of preliminary partial sequencing of thecDNA sequence from the polyprotein coding sequence of the virus Ljungan145 SL isolate. Said protein comprising said amino acid sequence SEQ IDNO: 4, said homologous sequences and said antigenic fragments are usefulas active ingredients in medicines and as diagnostic reagents indiagnostic kits.

A third aspect of the invention concerns an antiserum or antibodydirected against a structural protein of the virus defined in the firstaspect of the invention. An example of such a structural protein isdefined in the second aspect of the invention. Such an antiserum orantibody is useful as an active ingredient in medicines and asdiagnostic reagent in diagnostic kits. Both polyclonal and monoclonalantibodies may be used, and these are suitably produced by using saidvirus or fragments thereof specific for said virus for immunizingmammals.

A fourth aspect of the invention is directed to an antigen comprising atleast a part of a structural protein of the picornavirus defined in thefirst aspect of the invention, including a subunit thereof. An exampleof such an antigen is the protein and antigenic parts thereof defined inthe second aspect of the invention. Such an antigen of the invention isuseful as an active ingredient in medicines and as a diagnostic reagentin diagnostic kits.

A fifth aspect of the invention is directed to a diagnostic kitcomprising at least one member from the group consisting of

a) an antiserum or antibody according to the third aspect of theinvention or an antigen-binding part thereof,

b) an antigen according to the fourth aspect of the invention or anantibody-binding part thereof,

c) one or several probes designed with respect to the genome of thevirus according to the first aspect of the invention, and

d) one or several primers designed with respect to the genome of thevirus according to the first aspect of the invention.

The different members of a diagnostic kit will depend on the actualdiagnostic method to be used. In addition to the above-listed possiblemembers of the diagnostic kit, said kit may contain positive referencesamples, negative reference samples, diluents, washing solutions andbuffers as appropriate. The kit will further be accompanied byinstructions for use.

The above-listed members a) and b) find use in immunodiagnostic methods,such as enzyme-liked immunosorbent assay (ELISA), radioimmunoassay (RIA)or immunofluorescence assay (IFA).

The above-listed members c) and d) find use in direct virus detection.Preferably, a diagnostic method based on the PCR (polymer chainreaction) technique with such primers is utilized in the directdetection of a virus according to the invention.

All of the above mentioned diagnostic methods are well known in the art,and a man of ordinary skill in the art will readily select usefulmembers for a diagnostic kit in relation to the diagnostic method to beused.

A sixth aspect of the invention relates to a vaccine having as animmunizing or neutralizing component a member selected from the groupconsisting of

a) the virus according to the first aspect of the invention,

b) the virus according to the first aspect of the invention inattenuated form,

c) the virus according to the first aspect of the invention in killedform,

d) an antigen according to the fourth aspect of the invention, includinga subunit of the virus according to the first aspect of the invention,and

e) DNA corresponding to the genomic RNA of the virus according to thefirst aspect of the invention.

In an embodiment of this aspect of the invention said vaccine mayadditionally comprises an adjuvant. Such an adjuvant must of course bean adjuvant which is approved for use in vaccines by authoritiesresponsible for veterinary or human medicines.

The vaccine may contain other ingredients which are needed for specificpreparations intended for oral, subcutaneous, intramuscular orintradermal administration. Suitable additional ingredients aredisclosed in the European or US Pharmacopoeia.

The alternative members a), b) and c) are all examples of conventionalwhole virus, attenuated virus, and subunit vaccines developed for othertypes of viruses, and the member d) represents DNA incorporation intobody-specific cells, which then will express virus-specific structuresand elicit immunity against said virus.

A seventh aspect of the invention is directed to a picornavirusaccording to the first aspect of the invention, optionally in attenuatedor killed form, an antiserum or antibody according to the third aspectof the invention or an antigen according to the fourth aspect of theinvention, for use in a medicament (for veterinary or human use). Anexample of such a medicament is a vaccine according to the inventiondisclosed in the sixth aspect thereof.

The eight aspect of the invention concerns use of a picornavirusaccording to the first aspect of the invention, optionally in attenuatedor killed form, an antiserum or antibody according to the third aspectof the invention or an antigen according to the fourth aspect of theinvention, in the preparation of a medicament for prophylactic ortherapeutic treatment of a disease caused by said virus.

In an embodiment of said use the disease caused by said virus is one ofMyocarditis, Cardiomyopathia, Guillain Barré Syndrome, and DiabetesMellitus, Multiple Sclerosis, Chronic Fatigue Syndrome, MyastheniaGravis, Amyothrophic Lateral Sclerosis, Dermatomyositis, Polymyositis,Spontaneous Abortion, and Sudden Infant Death Syndrome.

A ninth aspect of the invention is directed to a method of prophylacticor therapeutic treatment of a disease caused by a virus according to thefirst aspect of the invention in a mammal, including human, whichcomprises administering to said mammal a prophylactically ortherapeutically effective amount of a medicament comprising as an activeingredient a member of the group consisting of

a) the virus according to the first aspect of the invention,

b) the virus according to the first aspect of the invention inattenuated form,

c) the virus according to the first aspect of the invention in killedform,

d) an antigen according to the fourth aspect of the invention, includinga subunit of the virus according to the first aspect of the invention,and

e) DNA corresponding to the genomic RNA of the virus according to thefirst aspect of the invention.

In an embodiment of said method the disease caused by said virus is oneof Myocarditis, Cardiomyopathia, Guillain Barré Syndrome, and DiabetesMellitus, Multiple Sclerosis, Chronic Fatigue Syndrome, MyastheniaGravis, Amyothrophic Lateral Sclerosis, Dermatomyositis, Polymyositis,Spontaneous Abortion, and Sudden Infant Death Syndrome.

The actual dosage regimen will be determined by the vaccine producerbased on animal experiments and clinical trials.

REFERENCES

1. Wesslen, L. et al Myocarditis caused by Chlamydia pneumonie (TWAR)and sudden unexpected death in a Swedish elite orienteer. The Lancet,340, 427-428 (1992).

2. Gard, S. Heller L. Hemagglutination by Col-MM-virus. Proc. Soc. Exp.Biol. Med. 76, 68-73 1951.

3. Francki, R. I. B., C. M. Fauquet, D. L. Knudson, and F. Brown (ed).1991. Classification and nomenclature of viruses. Fifth report of theInternational Committee in Taxonomy of viruses. Arch. Virol. 1991(Suppl. 2): 320-326.

4. Palmenberg, A. C. 1989. Sequence alignment of picornaviral capsidproteins, p. 211-241. In B. L. Semler and E. Ehrenfeld (ed.), Molecularaspects of picornavirus infection and detection. American Society forMicrobiology, Washington. D.C.

5. Stanway, G. 1990. Structure, function and evolution ofpicornaviruses. J. Gen. Virol. 71:2483-2501.

6. Lipton, H. L., A. Friedmann, P. Sethi, and J. R. Crowther. 1983.Characterization of Vilyuisk virus as a picornavirus. J. Med. Virol.12:195-203.

7. Zimmerman, J. J. Encephalomyocarditis. In: Handbook Series ofZoonoses. G. B. Beran (Ed.), CRC, Press Inc., Boca Raton, Fla. USA(1994).

8. Hubbard, G. B. et al. An encephalomyocarditis virus epizootic in ababoon colony. Lab. Anim. Sci. 42, 223-239 (1992).

9. Gaskin, J. M. et al. The tragedy of encephalomyocarditis virusinfection in zoological parks in Florida. Proc. Am. Assoc. Zoo. Vet 1-7(1980).

10. Craighead, J. E & McLane, M. F. Diabetes Mellitus: induction in miceby encephalomyocarditis virus. Science 162, 913-915 (1968).

11. Hayashi, K., Boucher, D. W. & Notkins, A. L. Virus induced diabetesmellitus. II. Relationship between -cell damage and hyperglycemia inmice infected with encephalomyocarditis virus. Am. J. Pathol. 75, 91-102(1974).

12. Dal Canto, M. C. Experimental models of virus-induced demyelination,in Handbook of Multiple Sclerosis, in Cook S D (ed), New York, MarcelDecker, pp 63-100 (1990).

13. Hirasawa, K., Han, J. Takeda, M., Itagaki, S. & Doi, K. J.Encephalomyocarditis (EMC) virus induced myocarditis by different virusvariants and mouse strains. Vet. Med. Sci. 54, 1125-1129 (1992).

14. Levin, R. et al. EMC virus infection in baboons as a model forstudies on antiviral substances. Antiviral Research 6, 277-283 (1986).

15. Blanchard, J. L., Soike, K. & Baskin, G. B. Encephalomyocarditisvirus infection in African green and squirrel monkeys: Comparison ofpathologic effect. Laboratory Animal Science 37, 635-639 (1987).

16. Lipton, H. L. & Dal Canto, M. C. Theiler's virus induceddemyelination: prevention by immunosuppression. Science 192, 62-64(1976).

17. Gajdusek, D. C. Encephalomyocarditis virus infection in childhood.Pediatrics 16, 902-906 (1955).

18. Niklasson, B. & Le Duc, J. Epidemiology of nephropathia epidemica inSweden. J. Inf. Dis. 155:269-276 (1987).

19. Riggs, J. L.: Immunofluorescent staining. In: Diagnostic proceduresfor Viral, Rickettsial, and Chlamydial infections, Am. Public. HealthAssoc., Washington 1979, 5th ed., p. 141.

20. Earley, E., Peralta, P. H. & Johnson, K. M.: A plaque neutralizationmethod for arboviruses. Proc. Soc. Exp. Biol. Med. 125:741. (1967)

1-14. (canceled)
 15. A Ljungan picornavirus comprising in a non-codingregion of the viral genome an RNA sequence corresponding to a cDNAsequence shown in Seq ID No:1 or a cDNA sequence having at least 75%sequence identity to Seq ID No:1. whereby said virus causes mammaliandisease.
 16. The virus according to claim 15, wherein said homologoussequences are selected from the group consisting essentially of Seq IDNO:2 and Seq ID NO:3.
 17. The virus according to claim 15 for use in amedicament for treating disease.
 18. The virus according to claim 17,wherein said medicament comprises at least a portion of said virus andis administered for treating disease.
 19. The virus according to claim18, wherein said virus is in a form selected from the group consistingessentially of attenuated, live, and killed.
 20. The virus according toclaim 15, wherein said cDNA sequences have at least 80% identity to SeqID No:1.
 21. The virus according to claim 15, wherein said cDNAsequences have at least 85% identity to Seq ID No:1.
 22. The virusaccording to claim 15, wherein said cDNA sequences have at least 90%identity to Seq ID No:1.
 23. A medicament for the treatment of disease,said medicament comprising the virus according to claim 15 in apharmaceutically acceptable carrier.
 24. The medicament according toclaim 23, wherein said medicament comprises at least a portion of saidvirus and is administered for treating disease.
 25. The medicamentaccording to claim 24, wherein said virus is in a form selected from thegroup consisting essentially of attenuated, live, and killed.
 26. Themedicament according to claim 23, further comprising an adjuvant. 27.The medicament according to claim 23, wherein said medicament is avaccine.
 28. Use of the Ljungan picornavirus according to claim 15 fortreating disease caused by said virus.
 29. A diagnostic kit comprisingat least one primer for priming a sequence that encodes a cDNA sequenceshown in Seq ID No:1 or a cDNA sequence having at least 75% sequenceidentity to Seq ID No:1.